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[Preprint]. 2024 May 4:2024.05.02.592248.
doi: 10.1101/2024.05.02.592248.

Sexually dimorphic phenotypes and the role of androgen receptors in UBE3A-dependent autism spectrum disorder

Yuan Tian  1 , Hui Qiao  1 , Ling-Qiang Zhu  2 , Heng-Ye Man  1   3   4
Affiliations

Sexually dimorphic phenotypes and the role of androgen receptors in UBE3A-dependent autism spectrum disorder

Yuan Tian et al. bioRxiv. .

Abstract

Autism spectrum disorders (ASDs) are characterized by social, communication, and behavioral challenges. UBE3A is one of the most common ASD genes. ASDs display a remarkable sex difference with a 4:1 male to female prevalence ratio; however, the underlying mechanism remains largely unknown. Using the UBE3A-overexpressing mouse model for ASD, we studied sex differences at behavioral, genetic, and molecular levels. We found that male mice with extra copies of Ube3A exhibited greater impairments in social interaction, repetitive self-grooming behavior, memory, and pain sensitivity, whereas female mice with UBE3A overexpression displayed greater olfactory defects. Social communication was impaired in both sexes, with males making more calls and females preferring complex syllables. At the molecular level, androgen receptor (AR) levels were reduced in both sexes due to enhanced degradation mediated by UBE3A. However, AR reduction significantly dysregulated AR target genes only in male, not female, UBE3A-overexpressing mice. Importantly, restoring AR levels in the brain effectively normalized the expression of AR target genes, and rescued the deficits in social preference, grooming behavior, and memory in male UBE3A-overexpressing mice, without affecting females. These findings suggest that AR and its signaling cascade play an essential role in mediating the sexually dimorphic changes in UBE3A-dependent ASD.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Fig. 1:
Fig. 1:. Sexually dimorphic deficits in social interaction, repetitive self-grooming behaviors, and ultrasonic vocalizations in male and female Ube3A 2xTg mice
(A,B) The paradigm for the three-chamber social test and traces of track paths. For social preference (A), an unfamiliar mouse was placed into either of the side chambers and the test mouse was allowed to move freely in the apparatus. For social novelty (B), a second mouse (novel mouse) was placed into the remaining empty chamber, and the test mouse was allowed to interact with both mice. (C,D) Quantification of the interaction time showed a decrease in preference for the stranger mouse (C) and novel mouse (D) in male Ube3A 2xTg mice, while female Ube3A 2xTg mice showed mild change in social preference (C) but clear impairment in social novelty (D). (E) Male but not female Ube3A 2xTg mice showed increased repetitive self-grooming behavior. (F) Representative vocalization recordings from P5 WT and Ube3A 2xTg mice. (G-K) Quantification of the number of calls (G), total call duration (H), mean call syllable duration (I), peak frequency (J), and peak amplitude (K) for P5 WT and Ube3A 2xTg mice. (L) Representative calls of each type used in syllable characterization. (M) Ube3A 2xTg female animals showed an increase in Complex type calls and a decrease in Simple type calls at P5, while Ube3A 2xTg males are similar to WT males. In (C), n=8 WT male; n=12 Tg male; n=9 WT female; n=9 Tg female. In (D), n=7 WT male; n=10 Tg male; n=8 WT female; n=9 Tg female. In (E), n=10 WT male; n=12 Tg male; n=10 WT female; n=12 Tg female. In (G-M) n=7 WT male; n=7 Tg male; n=7 WT female; n=6 Tg female. Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns, not significant. In (C), (D), (M) Three-way ANOVA with Bonferroni’s multiple comparisons test; In (E), (G-K) Two-way ANOVA with Bonferroni’s multiple comparisons test.
Fig. 2:
Fig. 2:. Sexually dimorphic deficits in memory, sensory functions, but not locomotion in male and female Ube3A 2xTg mice
(A) The paradigm for novel object recognition test. (B,C) Exploration time of familiar object vs. novel object 4 h (B) and 24 h (C) post training. Ube3A 2xTg animals displayed intact short time memory (B), and only Tg males showed impaired long-term memory (C). (D) The timeline of Barnes spatial memory maze. (E) Traces of track paths during the 5 d memory probe for WT and Ube3A 2xTg males. Blue arrows point to the escape hole. (F,G) Primary errors made to find the escape hole 24 h (F) and 5 d (G) after the last training. Ube3A 2xTg animals displayed intact short time memory (F), and only Tg males showed impaired long-term memory (G). (H-J) Both male and female Ube3A 2xTg mice showed decreased track lengths (H), normal mean velocities (I) and no change in relative time in center (J) in the open-field test. (K,L) Test mice were put on 55℃ hot plate and their latency to lick a hindpaw was recorded. Male Ube3A 2xTg mice showed reduced pain sensitivity compared to WT males, while Tg females were intact. (M) The paradigm for the Olfactory Detection Threshold Test. Test mice were exposed to increased concentration of vanilla. Time spent exploring vanilla vs. water was recorded. (N) Male Ube3A 2xTg mice showed intact olfactory sensitivity compared to WT males, whereas Tg females showed reduced olfactory sensitivity compared to WT females. In (B-C), n=6 WT male; n=11 Tg male; n=9 WT female; n=11 Tg female. In (F), n=6 WT male; n=10 Tg male; n=12 WT female; n=10 Tg female. In (G), n=6 WT male; n=10 Tg male; n=9 WT female; n=10 Tg female. In (H-J), n=10 WT male; n=11 Tg male; n=9 WT female; n=11 Tg female. In (K), n=10 WT male; n=12 Tg male; n=10 WT female; n=12 Tg female. In (N), n=10 WT male; n=10 Tg male; n=9 WT female; n=10 Tg female. Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns, not significant. In (B), (C), (N) Three-way ANOVA with Bonferroni’s multiple comparisons test; In (F-K) Two-way ANOVA with Bonferroni’s multiple comparisons test.
Fig. 3:
Fig. 3:. Androgen receptor expression is reduced in Ube3A 2xTg animals, leading to dysregulation of androgen responsive genes specifically in males
(A,B) Androgen receptors (AR) were reduced in both Tg male and Tg female prefrontal cortical lysates at P7. Estrogen receptors alpha (ERα) and beta (ERβ) didn’t change in Ube3A 2xTg animals compared to WT counterparts. n=4 animals/group. (C,D) Immunohistochemistry of WT and Tg cortical slices at P60 showed a marked decrease in AR immunointensity in Tg animals. n = 8 WT male; n = 10 Tg male; n = 10 WT female; n = 7 Tg female. Scale bars, 100 μm. (E,F) Top dysregulated androgen responsive genes (ARGs, E) and estrogen responsive genes (ERGs, F) in Ube3A 2xTg male and female brains, compared to their WT counterparts (n=3 animals/group). Blue fonts indicate dysregulation in male Ube3A 2xTg brains compared to WT males. Red fonts indicate dysregulation in female Ube3A 2xTg brains compared to WT females. Bold fonts indicate known SFARI ASD genes. ARGs were strongly dysregulated in Tg males but not Tg females. Little to no ERGs were dysregulated in Tg animals. Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001. ns, not significant. Two-way ANOVA with Bonferroni’s multiple comparisons test.
Fig. 4:
Fig. 4:. UBE3A overexpression can independently induce AR reduction by increasing ubiquitination and degradation of AR
(A) Representative images of immunocytochemistry for AR (red) at DIV 8 in rat cortical neuron cultures after transfected with GFP alone or together with UBE3A at DIV4. Scale bars, 25 μm. (B) UBE3A overexpression reduced AR intensity in primary culture neurons. GFP: n=22, GFP+UBE3A: n=19. (C,D) AR mRNA expression detected by qPCR was not altered in Tg males and Tg females at postnatal day 0 (C) and postnatal day 21 (D). P0: n=7 animals/group; P21: n=7 animals/group. (E) Degradation assay of AR with or without UBE3A. Transfected HEK cells were treated with cycloheximide (CHX) for various time points and cell lysates were collected to examine AR levels by Western blot. (F) Quantification of the degradation rate of AR over time; n=3 independent experiments. (G) AR ubiquitination assay. HEK293T cells were transfected with AR, HA-ubiquitin (Ubi), and either a vector control, UBE3A, or the E3 ligase dead mutant UBE3A C820A for 2 d. AR was immunoprecipitated and probed for ubiquitin. Cell lysates (input) were also probed to detect total protein levels. (H,I) AR ubiquitination assays using lysates of neurons infected with AAV2 GFP or AAV2 UBE3A virus for 10 d (H). Increased intensity of ubiquitination signals on AR was detected (I). n=3 independent experiments. (J,K) AR was immunoprecipitated from brain lysates and probed for ubiquitin signals. An elevated level in AR ubiquitination was detected in Ube3A 2xTg mice compared to WT. n=6 animals/group. Mean ± SEM. *p<0.05; **p<0.01. ns, not significant. In (C), (D), (F) Two-way ANOVA with Bonferroni’s multiple comparisons test; In (B), (I), (K) Unpaired two-tailed t test.
Fig. 5:
Fig. 5:. AR restoration in Ube3A 2xTg mouse brains rescues expression of androgen responsive genes in males, without affecting females
(A) Intracerebroventricular injection of GFP (LV-GFP) or GFP-fused AR (LV-GFP-AR) lentivirus at postnatal day 0. The mice were perfused at postnatal day 30 to 60 for cryostat to validify viral expression. LV-GFP-AR infection significantly increased AR expression in PFC and HPC. PFC, prefrontal cortex. HPC, hippocampus. Scale bars, 100 μm. (B-G) Expressions of six ASD-associated androgen responsive genes were rescued in Ube3A 2xTg males by ICV injection of LV-GFP-AR at postnatal day 0, detected by qPCR in postnatal day 40 prefrontal cortical lysates. WT male+LV-GFP: n=8, Tg male+LV-GFP: n=6, Tg male+LV-GFP-AR: n=6, WT female+LV-GFP: n=6, Tg female+LV-GFP: n=6, Tg female+LV-GFP-AR: n=6. Mean ± SEM. *p<0.05; **p<0.01; ns, not significant. Two-way ANOVA with Bonferroni’s multiple comparisons test.
Fig. 6:
Fig. 6:. AR restoration in Ube3A 2xTg mouse brains rescues social preference, memory, and repetitive self-grooming behaviors in males, without affecting females.
(A-D) Social behaviors in mice following ICV injection of LV-GFP-AR at postnatal day 0. Three chamber tests were performed at postnatal day 30 to 50. (A) Traces of track paths for social preference test. (B) LV-GFP-AR infection in Tg male rescued its preference for stranger mouse. Tg female showed normal social preference which was not affected by LV-GFP-AR infection. (C) Traces of track paths for social novelty test. (D) LV-GFP-AR infection failed to rescue impaired social novelty in Tg male and Tg female. WT male+LV-GFP: n=9, Tg male+LV-GFP: n=8, Tg male+LV-GFP-AR: n=12, WT female+LV-GFP: n=10, Tg female+LV-GFP: n=11, Tg female+LV-GFP-AR: n=8. (E) Long term memory in mice following ICV injection of LV-GFP-AR at postnatal day 0, detected by novel object recognition test at postnatal day 30 to 50. Tg males with LV-GFP-AR displayed better discrimination between the familiar vs. novel object compared to the LV-GFP infected Tg males, indicating successful rescue of memory. Tg female showed normal memory which was not affected by LV-GFP-AR. WT male+LV-GFP: n=9, Tg male+LV-GFP: n=8, Tg male+LV-GFP-AR: n=9, WT female+LV-GFP: n=10, Tg female+LV-GFP: n=9, Tg female+LV-GFP-AR: n=11. (F) Repetitive self-grooming behavior was impaired only in Tg males but not females. ICV injection of LV-GFP-AR successfully rescued grooming behaviors in Ube3A 2xTg males with no significant effect on Ube3A 2xTg females. WT male+LV-GFP: n=9, Tg male+LV-GFP: n=10, Tg male+LV-GFP-AR: n=12, WT female+LV-GFP: n=10, Tg female+LV-GFP: n=11, Tg female+LV-GFP-AR: n=11. Mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns, not significant. Two-way ANOVA with Bonferroni’s multiple comparisons test.
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